Process for producing (2&#39;-amino-2&#39;-deoxypentofuranosyl) guanine

ABSTRACT

The present invention relates to the production of a new compound (2&#39;-amino-2&#39;-deoxypentofuranosyl) guanine by a fermantation process using a microorganism belonging to the genus Aerobacter.

This is a division of application Ser. No. 516,708, filed Oct. 21, 1974,now U.S. Pat. No. 3,987,030, issued Oct. 19, 1976.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nuclear magnetic resonance spectrum of(2'-amino-2'-deoxypentofuranosyl) guanine.

FIG. 2 shows the ultra-violet absorption spectra of(2'-amino-2'-deoxypentofuranosyl) guanine.

FIG. 3 shows the infra-red absorption spectrum of(2'-amino-2'-deoxypentofuranosyl) guanine.

DESCRIPTION OF THE INVENTION

This invention relates to a new compound relating to nucleic acids. Moreparticularly, this invention relates to a new guanosine analoguedesignated as (2'-amino-2'-deoxypentofuranosyl) guanine.

The present invention is based upon the discovery that microorganismsbelonging to genus Aerobacter are capable of producing a new substancewhich exhibits similar characteristics to those of guanosine asdetermined by paper chromatography, UV absorption spectra and the like.After isolation and identification, it was confirmed that this substancewas a new guanosine analogue which may be used in analogous manner tothose of other guanosine analogues.

This invention also relates to a process for producing said newguanosine analogue by fermentation.

According to the present invention, a new compound represented by thefollowing general formula is obtained: ##STR1##

(2'-amino-2'-deoxypentofuranosyl) guanine (hereinafter designated as2'-APG) which is the new guanosine analogue according to the presentinvention can easily be isolated in the form of white plate-likecrystals having alkaline nature by concentrating, under reducedpressure, a solution of it in a suitable solvent such as, for example,water.

The new guanosine analogue exhibits the following maximum absorptions ofUV spectra, as shown in FIG. 2.

    ______________________________________                                        at (mμ)             in                                                     ______________________________________                                        253              neutral aqueous solution                                     256              0.1N HCl solution                                            258 & 266        0.1N NaOH solution                                           ______________________________________                                    

Rf values of the new guanosine analogue when determined by paperchromatography using No. 50 Filter Paper (available from Toyo RoshiKabushiki Kaisha, Japan) are as follows:

    ______________________________________                                        RF value        When developed by                                             ______________________________________                                        0.22       n-butanol:acetic acid:water (4:1:2)                                0.35       isopropanol:HCl (65:16.7)with addition                                        of water to give a total of 100                                    0.51       ethanol: 1 N ammonium acetate (75:30)                              ______________________________________                                    

As to the color reaction, the orcinol test and diphenylamine tests arenegative.

Elemental analysis, empirical formula and molecular weight of the newcompound are as follows:

Elemental analysis

C : 42.60 h : 4.95 n : 29.81

empirical formula C₁₀ H₁₄ N₆ O₄

Molecular weight 282.26

When the new compound is hydrolysed with 1N HCl for one hour in aboiling water bath and then subject to paper chromatography,aminopentose is observed as follows:

    ______________________________________                                        When developed by      position of Rf                                         ______________________________________                                        n-butanol:pyridine:water (6:4:3)                                                                     at 0.28                                                n-butanol:ethyl acetate: water (7:1:2)                                                               0.42                                                   n-butanol:acetic acid: water (4:1:2)                                                                 0.31                                                   ______________________________________                                    

This aminopentose is colored as follows, thereby confirming it as2'-aminopentose.

    ______________________________________                                        colour               by                                                       ______________________________________                                        pink           p-anisidine reagent                                            purple         ninhydrin reagent                                              pink           Elson-Morgan's procedure                                       blue           Tsuji-Kinoshita reaction                                       ______________________________________                                    

In addition, results obtained by IR spectrum and nuclear magneticresonance also serve to identify the new compound of the presentinvention.

The new compound of the present invention has excellent physiologicalactivity and does not exhibit any toxity for example when administeredto the abdominal cavity of DD-type mouse in an amount of 800 mg per Kgof mouse. Anti-cancer activity is observed when this new compound isadministered intravenously in an amount of 120mg/Kg per day continuouslyfor 8 days to a mouse which has been transplanted with Salcoma 180solid-type cancer, 10 mcg/ml of this new compound inhibits the growth ofcancer cells HeLa S₃ in vitro.

400 mg of crystalline 2'-APG is hydrolysed with 1N HCl at a temperatureof 100° C for 1 hour, and passed through Dowex 50W (H⁺ form, tradenamefor an ion exchange resin, available from Dowex Chemical Inc., USA). Thesugar fraction of the 2'-APG is isolated as hydrochloride which is thencrystallized with a methanol-acetone solution to give 100 mg ofcrystalline product. The decomposition temperature of the crystallineproduct is 143°-150° C and its angle of optical rotation is [α]_(D) ²⁷= + 13°4 (initial, extra-polated) → -3.5° (C = 0.75, H₂ O). By comparingthe results obtained with the standard values of synthesized2-aminopentose, it is found that the sugar part of the crystalline2'-APG is D-2-amino-2-deoxyribose. 2'-APG according to the presentinvention has an angle of optical rotation of [α]_(D) ²⁶ = -56.6° (C =0.5, H₂ O). On the other hand, the angle of optical rotation of theguanosine (9-β-ribofuranosyl guanine) is [α]_(D) ²⁶ = -72° (C = 1.4,0.1N NaOH). The angles of optical rotation of adenosines which areanalogous compounds to 2'-APG of the present invention are as follows:

i. 9-β-adenosine

[α]_(D) ¹¹ = -61.7° (C = 0.706, H₂ O)

ii. 9-α-2'-amino-2'-deoxyadenosine

[α]_(D) ²³ = +90° ± 2° (C = 0.635, methanol)

iii. 9-β-2'-amino-2'-deoxyadenosine

[α]_(D) ²² = -66° ± 2° (C = 0.98, methanol)

From the above results, it is confirmed that 2'-APG of the presentinvention has a β -conformation.

The present invention relates further to a process for producing a newguanosine analogue by fermentation.

Any and all micoorganisms which belong to the genus Aerobacter(Enterobacter) may be used for the process of the present invention. Thespecies of microorganism which has been found especially useful for theprocess of the present invention is Aerobacter cloacae (Enterobactercloacae) (FERM-P No. 1893).

The microorganisms referred to in the present specification are freelyavailable to the public from the Fermantation Research Institute, Agencyof Industrial and Technology, Japan.

Carbon sources which may be used for the culture medium used in thepresent invention include carbohydrates such as glucose, sucrose,starch, sorbitol etc., alcohols, and organic acids such as acetic acid,etc.

Suitable nitrogen sources include, for example, inorganic nitrogencompounds such as ammonium sulphate, etc. and organic nitrogen compoundssuch as meat extract, yeast extract, peptone, etc.

In carrying out the process of the present invention, the culture mediumcontaining the above-mentioned carbon source, nitrogen source, inorganiccompounds such as phosphate, sulphate, hydrochloride, metallic salt suchas iron, mananese, magnesium, potassium, sodium, etc. andgrowth-promoting factors is sterilized and then inoculated with theaforesaid microorganism for aerobic culturing. It is possible to improvethe yield of the new compound by adding purine nucleotides such asinosinic acid, xanthylic acid, guanylic acid, etc. or its precursor tothe medium.

The cultivation may be effected at a temperature of from 20° to 40° Cand a pH of from 5 to 8, preferably from 6 to 7. In this case, the pH isadjusted by the addition of urea solution, aqueous ammonia or ammoniumcarbonate. The cultivation is completed when the amount of 2'-APGproduced reaches a maximum period of from 1 to 7 days is usuallysufficient for effecting cultivation.

When isolating the 2'-APG produced, cell bodies are removed from theculture medium by means of centrifugation or filtration. The 2'-APG isthen recovered from the culture liquor by passing the liquor through anacidic ion exchange resin to selectively absorb the 2'-APG thereon. Ifnecessary, absorbing agents may be used. The 2'-APG is eluted using, forexample, water or aqueous ammonia and the eluate is concentrated underreduced pressure to produce purified crystalline 2'-APG.

The following non-limitative examples further illustrate the presentinvention.

EXAMPLE 1

Aerobacter cloacae (Enterobacter cloacae) (FERM-P No. 1893) wasinoculated as a seed in a seed medium composed of meat extract (1.0%),peptone (1.0%), yeast extract (0.3%), table salt (0.3%) and sorbitol(2.0%). The pH of the seed medium was adjusted to 7.3 beforesterilization, and the medium cultured at a temperature of 30° C for 24hours with shaking. The resultant seed was put into a 2-liter Florenceflask containing a fermentation medium having the following composition.

    ______________________________________                                        sucroe      10.0%      yeast extract                                                                             0.1%                                       ammonium sulphate                                                                         1.0%       KCl         0.8%                                       M.sub.g SO.sub.4                                                                          0.1%       K.sub.2 HPO.sub.4                                                                         0.02%                                      F.sub.e SO.sub.4                                                                          40 mg/l    ZnSO.sub.4  3 mg/l                                     ______________________________________                                    

The pH of the fermentation medium was adjusted to 7.0 beforesterilization and the cultivation was carried out at 30° C for 24 hoursusing a rotary shaking method to yield 205 mg of 2'-APG per liter of thefermented liquor which was then centrifuged to obtain the supernatantsolution. The supernatant (3 liters) was passed through a resin columnpacked with Amberlite IRC-50 (tradename for a weakly acidic ion exchangeresin in H-form, available from Rohm & Haas, U.S.A.) and eluted with0.5N aqueous ammonia. It was then passed through a resin column packedwith Diaion SK 1B (tradename for a strongly acidic ion exchange resin inNH₄ -form, available from Mitsubishi Kasei Kogyo Kabushiki Kaisha,Japan) and eluted with 10mM aqueous ammonia. After concentration, thesolution was made up with 10mM aqueous ammonia and was passed through acolumn packed with Diaion HP (tradename for a synthetic absorbing agent,available from Mitsubishi Kasei Kogyo Kabushiki Kaisha, Japan). Theeluted fraction containing 2'-APG were collected, concentrated and thenacetone added (10 times the quantity of the fractions). The supernatantwas concentrated to obtain crystals of 2'-APG (440 mg) having a meltingpoint of 252°-254° C (decomp.).

EXAMPLE 2

The cultivation was carried out in an analogous manner to that describedin Example 1 with the exception that sodium inosinate (0.5 g/l) wasadded to the fermentation medium. There were obtained 660 mg of 2'-APGper liter of the fermented liquor.

EXAMPLE 3

The seed culture was prepared in an analogous manner to that describedin Example 1 with the exception that the seed was cultured for 15 hoursand then transferred to a 30-liter jar fermentor containing 18 liters ofthe fermentation medium having the same composition as that described inExample 1. The ratio of inoculation was 5% based upon the quantity ofthe fermentation medium. During the fermentation which was carried outat a temperature of 30° C for 24 hours with shaking (350 r.p.m.) andaeration (one liter of sterilized air per 1 liter of the medium), the pHof the medium was controlled to 6.5 to 7.0 with aqueous ammonia. Aftercompletion of the cultivation, the fermented liquor contained 300 mg/lof 2'-APG. By treating the fermented liquor in an analogous manner tothat described in Example 1, there were obtained crystals of 2'-APG(2.9g).

EXAMPLE 4

The cultivation was carried out in an analogous manner to that describedin Example 3 with the exception that xanthylic acid (sodium salt) wasadded to the fermentation medium to give a concentration of 1 g/l. Aftercompletion of the fermentation there was obtained 800 mg of 2'-APG perliter of the fermented liquor which was then treated in an analogousmanner to that described in Example 1 to give 9.2 g of crystals of2'-APG.

Having described the present invention, that which is sought to beprotected is set forth in the following claims.

We claim:
 1. A process for producing (2'-amino-2'-deoxypentofuranosyl)guanine, comprising culturing a microorganism capable of producing(2'-amino-2'-deoxypentofuranosyl) guanine and belonging to the genusAerobacter in a culture medium, and recovering the accumulated(2'-amino-2'-deoxypentofuranosyl) guanine from the fermented liquor. 2.The process of claim 1 wherein the microorganism is one belonging to thespecies of Aerobacter cloacae.
 3. The process of claim 2 wherein themicroorganism is Aerobacter cloacae (FERM-P No. 1893).
 4. The process ofclaim 1 wherein culturing is effected at a temperature of from 20° to40° C and at a pH of 5 to
 8. 5. The process of claim 4 wherein theculture medium contains a carbon source, a nitrogen source and inorganiccompounds.
 6. The process of claim 4 where a pH of 6 to 7 is employedduring cultivation.
 7. The process of claim 1 wherein after cultivation,the fermented liquid is centrifuged and the supernatant liquid passedthrough a series of ion exchange resin column with intermittent elutionthereof with aqueous ammonia in order to obtain a fraction rich in thedesired product.
 8. The process of claim 7 wherein said fraction istreated with acetone and the supernatant liquid concentrated to givecrystals of (2'-amino-2'-deoxypentofuranosyl) guanine.